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Satellite DNA of the red flour beetle Tribolium castaneum--comparative study of satellites from the genus Tribolium.

A highly abundant satellite DNA comprising 17% of the Tribolium castaneum (Insecta, Coleoptera) genome was cloned and sequenced. The satellite monomer is 360 bp long, has a high A+T content of 73%, and lacks significant internal substructures. The sequence variability is 3.6%, essentially due to random distribution of single-point mutations. The satellite is evenly distributed in the regions of centromeric heterochromatin of all 20 chromosomes, as shown by fluorescent in situ hybridization. Comparison of T. castaneum satellite with those from three different but congeneric species reveals the highest sequence similarity of 47.1% with the satellite from the sibling species Tribolium freemani. The phylogenetic relationships among Tribolium species deduced from satellite sequence agree with those based on karyological, chemotaxonomic, and hybridization data. This indicates a parallel in the divergence of satellites and some genetic and cytogenetic characters. Despite low mutual sequence similarity, which makes them species-specific, Tribolium satellites have a common structural characteristic: a block of about 95% A+T content, 20 to 42 bp long, flanked at one side by an inverted repeat which can potentially form a thermodynamically stable dyad structure. Since similar structural features are found in centromeric DNA of Saccharomyces cerevisiae and Chironomus pallidivittatus, their possible importance in centromere function may be inferred.

Animals↗

Inter- and intraspecific sexual discrimination in the flour beetles Tribolium castaneum and Tribolium confusum.

In Tribolium castaneum (CS) and T. confusum (CF), intra- and interspecific rates of homosexual mounting have been measured. The intraspecific results are compatible with the hypothesis of both species being sexually indiscriminate. However, the CF intraspecific rates were very high (35%-53% of mountings were homosexual), suggesting a lower sexual attractiveness, or a stronger rejection to being mounted, of CF females relative to conspecific males. CS males discriminate between species but, in interspecific contacts, preferentially mounted CF males rather than CF females. CF males do not discriminate between species, but the loss of sexual attractiveness of CF females, or their rejection to being mounted, may act as a precopulatory isolation mechanism.

Animals↗

Benzoquinones of the beetles, Tribolium castaneum and Tribolium confusum.

Tribolium castaneum and T. confusum were washed in HPLC-grade methanol, and the methanolic washes were analyzed by UV spectroscopy, reversed phase HPLC, and GC/MS. The methanolic washes from both species contained methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ). The amounts of MBQ recovered from the two species were not significantly different, but the amounts of EBQ and total benzoquinones (MBQ+EBQ) recovered from T. castaneum were significantly greater than for those recovered from T. confusum. The methods described are superior to previous methods for isolating, identifying, and quantifying the benzoquinones in these beetles, since they are relatively simple, fast, do not require handling of the beetles, and are sensitive enough to quantify the benzoquinones of a single beetle.

Animals↗

Quantification of benzoquinones in the flour beetles. Tribolium castaneum and Tribolium confusum.

Benzoquinones were extracted from flour beetles by rinsing them for 8 min in methanol followed by homogenization in fresh methanol. This procedure recovered > 96% of the total benzoquinones from both species, with the methanolic rinse and supernatant representing the "surface" and "total" benzoquinones. As determined by UV/VIS spectrometry and reversed-phase HPLC, the methanolic rinse contained methyl- and ethyl-1,4-benzoquinone (MBQ and EBQ), but no substituted hydroquinones, The methanolic supernatant contained MBQ and EBQ, and methyl- and ethyl-hydroquinone. When stored at -20 degrees C for 72 hr, the chemical composition of the methanolic rinse did not change, but the composition of the methanolic homogenate did change. Notably, the amounts of benzoquinones in the methanolic supernatant decreased during storage. The method described represents an improvement over those described previously in that it uses less hazardous solvents for extraction (methanol vs more non-polar solvents such as hexane) and allows for the differentiation and quantification of the surface and total benzoquinones of these beetles.

Animals↗

Proboscipedia represses distal signaling in the embryonic gnathal limb fields of Tribolium castaneum.

Orthologs of the Hox genes Sex combs reduced ( Scr) and proboscipedia ( pd) are active in the developing labial appendages of all insect species tested. The remarkable variation among insect gnathal structures, particularly in the distal podomeres, suggests two Hox genes may enhance the adaptive potential of gnathal appendage morphology. Functional studies in the fruitfly Drosophila melanogaster, the flour beetle Tribolium castaneum and the milkweed bug Oncopeltus fasciatus show that cooperation between Scr and pb has been generally conserved, but specific mechanisms have been altered during evolution. Cross-regulation of pb by Scr is evident in Drosophila and Tribolium, the more closely related of the three species, but not in Oncopeltus. In all three species, pb function is restricted to the distal podomeres, but details are only known for Drosophila and Oncopeltus, two species exhibiting specialized stylate-haustellate mouthparts. Drosophila pb is required for distal Scr expression, and to repress the appendage patterning genes dachshund and Distal-less ( Dll). Oncopeltus pb has the novel capacity to specify leg fates. Little is known about distal functions of Tribolium pb. Hypomorphic mutations of the Tribolium pb ortholog maxillopedia can be arranged in a graded phenotypic series of palp to leg transformations along both the proximodistal and dorsoventral axes. Mid-embryonic expression profiles of Tribolium pb, Scr, wingless ( wg) and Dll genes were examined in maxillopedia hypomorphic and null mutant backgrounds. Levels of pb and Scr are significantly reduced in the distal appendage field. Tribolium pb therefore positively regulates distal Scr expression, a role that it has in common with Drosophila pb. Tribolium wg is normally down-regulated in the distal domain of the embryonic gnathal appendage buds. It becomes activated distally in maxillopedia hypomorphs. Repression of wg by pb has not been reported in the labial imaginal discs of Drosophila. Alterations of Tribolium Scr and wg expression occur in Dll-expressing cells, however, unlike in Drosophila labial imaginal discs, Dll expression appears unaffected in pb hypomorphic backgrounds. We conclude that the Hox genes Sex combs reduced and proboscipedia control an appendage organizer and cell autonomous fate determination during embryonic labial palp development in Tribolium.

Animals↗

Maxillopedia is the Tribolium ortholog of proboscipedia.

Null mutations in the Drosophila melanogaster homeotic gene proboscipedia (pb) cause transformation of the adult labial palps to legs. The similar phenotype produced by mutations in the Tribolium castaneum homeotic complex (HOMC) gene maxillopedia (mxp) has led to suggestions that the two genes may be orthologous. We have cloned the Tribolium ortholog of pb, which predicts a protein with a homeodomain identical to that of Drosophila Pb. The two proteins also share several additional regions of identity, including an N-box, a motif unique to Pb orthologs. We have identified a frameshift mutation within Tribolium pb associated with an mxp null mutation, demonstrating that Tribolium pb corresponds to the mxp genetic locus. Thus, we will refer to the cloned gene as mxp. In addition, we have begun to construct a molecular map of the Tribolium HOMC. Two overlapping BAC clones which span the mxp locus also include the Tribolium labial ortholog (Tclabial) and part of Tczerknüllt, indicating that the order of these genes in the HOMC is conserved between Drosophila and Tribolium.

Amino Acid Sequence↗

A comparison of homologous developmental genes from Drosophila and Tribolium reveals major differences in length and trinucleotide repeat content.

The flour beetle Tribolium castaneum has become an important model organism for comparative studies of insect development. Many developmentally important genes have now been cloned from both Tribolium and Drosophila and their expression characteristics were studied. We analyze here the complete coding sequences of 17 homologous gene pairs from D. melanogaster and T. castaneum, most of which encode transcription factors. We find that the Tribolium genes are on average 30% shorter than their Drosophila homologues. This appears to be due largely to the almost-complete absence of trinucleotide repeats in the coding sequences of Tribolium as well as the generally lower degree of internal repetitiveness. Clusters of polar and other amino acids such as glutamine, proline, and serine, which are often considered to be important for transcriptional activation domains in Drosophila, are almost completely absent in Tribolium. Codon usage is generally less biased in Tribolium, although we find a similar tendency for the preference of G- or C-ending codons and a higher bias in conserved subregions of the proteins as in Drosophila. Most of the aminoacid substitutions in the DNA-binding domains of the transcription factors occur at residues that do not make a specific contact to DNA, suggesting that the recognition sequences are likely to be conserved between the two species.

Amino Acid Sequence↗

Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function.

Segmentation is well understood in Drosophila, where all segments are determined at the blastoderm stage. In the flour beetle Tribolium castaneum, as in most insects, the posterior segments are added at later stages from a posteriorly located growth zone, suggesting that formation of these segments may rely on a different mechanism. Nevertheless, the expression and function of many segmentation genes seem conserved between Tribolium and Drosophila. We have cloned the Tribolium ortholog of the abdominal gap gene giant. As in Drosophila, Tribolium giant is expressed in two primary domains, one each in the head and trunk. Although the position of the anterior domain is conserved, the posterior domain is located at least four segments anterior to that of Drosophila. Knockdown phenotypes generated with morpholino oligonucleotides, as well as embryonic and parental RNA interference, indicate that giant is required for segment formation and identity also in Tribolium. In giant-depleted embryos, the maxillary and labial segment primordia are normally formed but assume thoracic identity. The segmentation process is disrupted only in postgnathal metamers. Unlike Drosophila, segmentation defects are not restricted to a limited domain but extend to all thoracic and abdominal segments, many of which are specified long after giant expression has ceased. These data show that giant in Tribolium does not function as in Drosophila, and suggest that posterior gap genes underwent major regulatory and functional changes during the evolution from short to long germ embryogenesis.

Abdomen↗

Regulation of the Tribolium homologues of caudal and hunchback in Drosophila: evidence for maternal gradient systems in a short germ embryo.

In short germ embryos, the germ rudiment forms at the posterior ventral side of the egg, while the anterior-dorsal region becomes the extra-embryonic serosa. It is difficult to see how an anterior gradient like that of bicoid in Drosophila could in these embryos be directly involved in patterning of the germ rudiment. Moreover, since it has not yet been possible to recover a bicoid homologue from any species outside the diptera, it has been speculated that the anterior bicoid gradient could be a late addition during insect evolution. We addressed this question by analysing the regulation of potential target genes of bicoid in the short germ embryo of Tribolium castaneum. We demonstrate that homologues of caudal and hunchback from Tribolium are regulated by Drosophila bicoid. In Drosophila, maternal caudal RNA is translationally repressed by bicoid. We find that Tribolium caudal RNA is also translationally repressed by bicoid, when it is transferred into Drosophila embryos under a maternal promoter. This strongly suggests that a functional bicoid homologue must exist in Tribolium. The second target gene, hunchback, is transcriptionally activated by bicoid in Drosophila. Transfer of the regulatory region of Tribolium hunchback into Drosophila also results in regulation by early maternal factors, including bicoid, but in a pattern that is more reminiscent of Tribolium hunchback expression, namely in two early blastoderm domains. Using enhancer mapping constructs and footprinting, we show that caudal activates the posterior of these domains via a specific promoter. Our experiments suggest that a major event in the evolutionary transition from short to long germ embryogenesis was the switch from activation of the hunchback gap domain by caudal to direct activation by bicoid. This regulatory switch can explain how this domain shifted from a posterior location in short germ embryos to its anterior position in long germ insects, and it also suggest how an anterior gradient can pattern the germ rudiment in short germ embryos, i.e. by regulating the expression of caudal.

Animals↗

Multiple origins of cyclodiene insecticide resistance in Tribolium castaneum (Coleoptera: Tenebrionidae).

The number of origins of pesticide resistance-associated mutations is important not only to our understanding of the evolution of resistance but also in modeling its spread. Previous studies of amplified esterase genes in a highly dispersive Culex mosquito have suggested that insecticide resistance-associated mutations (specifically a single-gene duplication event) can occur a single time and then spread throughout global populations. In order to provide data for resistance-associated point mutations, which are more typical of pesticide mechanisms as a whole, we studied the number of independent origins of cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum. Target-site insensitivity to cyclodienes is conferred by single point mutations in the gene Resistance to dieldrin (Rdl), which codes for a subunit of a gamma-aminobutyric acid (GABA) receptor. These point mutations are associated with replacements of alanine 302 which render the receptor insensitive to block by the insecticide. We collected 141 strains of Tribolium worldwide and screened them for resistance. Twenty-four strains contained resistant individuals. After homozygosing 23 of these resistance alleles we derived a nucleotide sequence phylogeny of the resistant strains from a 694-bp section of Rdl, encompassing exon 7 (which contains the resistance-associated mutation) and part of a flanking intron. The phylogeny also included six susceptible alleles chosen at random from a range of geographical locations. Resistance alleles fell into six clades and three clades contained both resistant and susceptible alleles. Although statistical analysis provided support at only the 5-6% level, the pattern of variation in resistance alleles is more readily explained by multiple independent origins of resistance than by spread of a single resistance-associated mutation. For example, two resistance alleles differed from two susceptible alleles only by the resistance-associated mutation itself, suggesting that they form the susceptible ancestors and that resistance arose independently in several susceptible backgrounds. This suggests that in Tribolium Rdl, de novo mutations for resistance have arisen independently in several populations. Identical alleles were found in geographically distant regions as well, also implying that some Rdl alleles have been exported in stored grain. These differences from the Culex study may stem both from differences in the population genetics of Tribolium versus that of mosquitoes and differences in mutation rates associated with point mutations versus gene duplication events. The Tribolium data therefore suggest that multiple origins of insecticide resistance (associated with specific point mutations) may be more common than the spread of single events. These findings have implications for the way in which we model the evolution and spread of insecticide resistance genes and also suggest that parallel adaptive substitutions may not be uncommon in phyletic evolution.

Animals↗

Interaction between radiation effects, gravity and other environmental factors in Tribolium confusum.

Multicellular organisms possess homeostatic control systems, which, in responding to changes of the external environment, modify the internal milieu of the organism accordingly, in order to make survival and normal physiological processes possible. This group has studied effects on physiological processes of single and combined environmental factors in the flour beetle, Tribolium confusum. Studies included low- and high-LET radiation, gravity compensation, near-weightlessness in space flight, ambient temperature, atmospheric composition, and magnetic field effects on the growth and development of Tribolium. For somatic effects, there appears to be a "normal physiological range" for each of these environmental variables; moreover, their effects seem independent of each other. When one of the listed environmental factors is near the limits of normal physiological tolerance, however, marked synergism has been observed between the effects of this factor and other environmental stresses. Fertilized Tribolium eggs showed a differential radiosensitivity to external irradiation, and a linear, dose-effect relationship was obtained, as accelerated heavy nitrogen and oxygen ions were used. Synergism was found when Tribolium were irradiated with ionizing radiation and subsequently exposed to temperature either below or above their normal physiological range. Gravity compensation or near-weightlessness in space flight have adverse effects on the development of Tribolium when an ambient temperature is near the higher or lower limit of tolerance. Similar results were observed when pupae were exposed to a combination of oxygen, temperature and magnetic field stresses.

Animals↗

Conserved patterns in the evolution of Tribolium satellite DNAs.

Two satellite DNAs, TANAPH and TDEST, isolated from the beetle species Tribolium anaphe and Tribolium destructor, respectively, are characterized and compared with previously described Tribolium satellites, in order to deduce possible constraints on satellite sequence evolution between closely related species. Sequence diversity analysis of cloned monomers reveals the presence of variable and conserved segments in both satellites. In addition, non-random organization of As or Ts and their periodical distribution in the form of A or T >/=3 tracts, as well as CENP-B box-like motifs and dyad structures have been found in both satellites. Similar structural features are also present in satellites from other Tribolium species. We therefore propose that they, together with the observed non-constant rate of evolution along the satellite sequence, could be related to putative protein binding sites and suggest a possible selective pressure affecting these sequences. Tribolium satellites, including TANAPH and TDEST, are located in the pericentromeric heterochromatin of all chromosomes of the corresponding species. Since satellites from different species exhibit no significant sequence homology, we propose that they did not originate from a common ancestral sequence. More probably, they derive from simple sequence modules some of which could represent protein binding sites. Shuffling of simple sequence modules could generate different satellites, able to perform a similar role in different species.

Animals↗